Tetrazolium compounds (tetrazolium salts) have been utilized for the quantitative analysis of dehydrogenases such as lactate dehydrogenase (hereinafter abbreviated to “LDH”), alcohol dehydrogenase and glutamate dehydrogenase. This is because a tetrazolium compound receives a hydrogen liberated by the action of a dehydrogenase, among various types as mentioned above, via an intermediate electron carrier to form a formazan, in which the measurement of the absorbance of the resultant formazan enables the quantitative analysis of the dehydrogenase or the substrate thereof.
Among these dehydrogenases, in particular, LDH is distributed in all somatic cells, and abundantly exists particularly in myocardia, liver and skeletal muscles and it is known that serum LDH activity markedly increases in patients with diseases such as cardiac infarction, malignant tumor, hepatic disease, progressive muscular atrophy, intravascular hemolysis and megaloblastic anemia. Therefore, measurement of LDH activity in blood provides meaningful information for clinical diagnosis. There is also desired a method based on dehydrogenases that experience little interference from biological materials, for measurements of uric acid and bile acid in blood.
Determination of cell proliferations and cytotoxicities has recently been made possible by using dehydrogenase activity in cultured cells or leaked out of cells, as an index, and the method is widely utilized as a convenient measure to learn the toxicities of chemicals and the efficacies of newly developed therapeutic products.
As the hydrogen receptors for the above-mentioned purposes, there have been generally utilized such materials as 3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis-[2-(4-nitrophenyl)-5-2H tetrazolium chloride] (hereinafter abbreviated to “nitro-TB”) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (hereinafter abbreviated to “MTT”).
Formazan formed from nitro-TB or MTT upon receiving hydrogen is, however, insoluble in water and inconvenient for use. A drawback encountered particularly in the application of these compounds to the automated analysis or microplate analysis is that the formazan formed stains the detection system such as cell, tubes and microplate. The use of a tetrazolium salt that will form a water-soluble formazan is therefore required to eliminate the drawback. Especially, in measuring a biological sample, a purple to blue formazan with absorption at a longer wavelength is desired so as to avoid interference with the color of biological material which may be included in the sample.
The research group to which the present inventors belong previously developed tetrazolium compounds that fulfill these requirements (JP-B-2592436, JP-B-2819258 and JP-B-2995880). While the tetrazolium compounds disclosed in these patents serve as reagents that are practically applicable to automated or microplate analysis of dehydrogenases and the substrates thereof, more improvement is needed. In particular, the water-soluble tetrazolium compounds proposed so far that form the formazans with long-wavelength absorption are unstable in aqueous solution, which makes long-term storage as reagents difficult.